Permeabilized cardiomyocytes have intracellular structures that restrict the diffusion of ADP from the surrounding medium to the mitochondria. Intracellular diffusion restrictions are also present in rainbow trout (Oncorhynchus mykiss) cardiomyocytes. This was surprising, because trout cardiomyocytes are no more than a few μm thick, lack t-tubules, have a lower sarcoplasmic reticulum density and only a single layer of myofilaments surrounding a central core of mitochondria. However, the extent of diffusion restrictions is smaller than in mammalian cardiomyocytes. The aim of the present study was to locate the diffusion restrictions in permeabilized trout cardiomyocytes. We measured mitochondrial respiration stimulated by ADP and ATP, and evaluated the rate of inhibition of ATP stimulated respiration by an ADP trapping system, consisting of phosphoenolpyruvate (PEP) activating endogenous and exogenous pyruvate kinase (PK), which competes with mitochondria for ADP. We found a high activity of hexokinase, which stimulates mitochondrial respiration when activated by glucose. To study its role in more detail, all experiments were performed in the absence and presence of 2 mM glucose. Additionally, we performed ADP titrations on cells alone, in the presence of glucose, and in the presence of creatine kinase and creatine. Our results showed that in contrast to rat cardiomyocytes, there is no functional coupling between creatine kinase and respiration. Trout cardiomyocytes have a high activity of hexokinase, which is functionally coupled to respiration. This suggests that trout cardiomyocytes have a different ADP-feedback system than mammalian cardiomyocytes. The experimental results were fitted with a mathematical model showing that diffusion is restricted in cytosol as well as by the outer mitochondrial membrane.